Most human genes require pre-mRNA splicing for expression, and >15% of the mutations that cause human genetic diseases disrupt splicing. A long term goal of our research is to understand the rules for RNA splicing generally. This proposal seeks to identify the sequence-specific splicing regulatory proteins that bind to exonic splicing silencers (ESSs) and related elements, and to characterize their functions and regulatory targets. In Phase 1 of the project, a system for efficient identification of trans-acting protein factors that act through specific ESS elements will be developed and applied to identify factors for a significant proportion of the FAS- ESS motifs identified previously by our lab. Phase 2 will seek improved understanding of the functions of selected splicing regulators by identifying the exons and transcripts they regulate. In phase 3, we will characterize the binding specificity and identify direct regulatory targets of a subset of these factors using technologies to assess their transcriptome-wide binding locations. A combination of RNAi-based screening, crosslinking and immunoprecipitation, high-throughput sequencing and computational analyses will be used. These studies will enable improved prediction of the consequences of genetic mutations or polymorphisms that alter splicing regulatory elements and will identify potential therapeutic targets in these cases. Public Health Relevance Statement: This project seeks to identify and characterize sequence-specific splicing regulatory proteins in human cells;such factors play important roles in both constitutive and alternative splicing of human genes. Alternative splicing is a gene regulatory mechanism that is used by more than half of all human genes, and genetic mutations or polymorphisms that disrupt splicing are a very common contributor to human disease. Identifying novel splicing regulatory factors and characterizing the specificity and targets of known regulatory factors will improve our ability to predict which genetic variations will have splicing phenotypes, and the factors involved. Knowing the protein factor that recognizes a particular cis-element such as an exonic splicing silencer identifies a potential therapeutic target in cases where a disease results from activity of a cis-element created by a mutation or polymorphism.